Isovaleryl CoA dehydrogenase, an enzyme involved in leucine metabolism, will be isolated from rat liver mitochondria and used to transform stereospecifically labeled (3S)-[4-13C]-isovaleryl CoA to Beta-methylcrotonyl CoA. The results will reveal the stereochemistry of the dehydrogenation reaction. The absolute steric course and stereospecificity of ketopantoate hydroxymethyl-transferase (KHMT), which catalyzes the first step of pantothenic acid and coenzyme A biosynthesis, will be determined, using isolated KHMT in a coupled assay with ketopantoate reductase. The steric course of deoxycytidylate hydroxymethylase, a methylenetetrahydrofolate-dependent enzyme induced by T-even bacteriophages in E. coli, will be determined. The mechanism of 4'-phosphopantothenoyl-L-cysteine decarboxylase, another enzyme involved in the biosynthesis of the important acyl group carrier coenzyme A, will be investigated. In particular, the active site groups responsible for reported inhibitions by pyridoxal phosphate and by carbonyl-reactive reagents will be identified.